| zeolite assisted refolding |
| n/a |
| 20 mM Tris–HCl [pH 7.5], 500 mM NaCl, and 6 M Gdn-HCl) containing 50 mM DTT |
| 50 mM Tris–HCl [pH 8.5], 500 mM NaCl, 5 mg/mL PEG 20000, 1 mM EDTA, 10 mM Cys, and 1 mM cystine) containing Gdn-HCl and/or Arg |
| not specified |
| no tag |
| 8.5 |
| 4.0 |
| n/a |
| n/a |
| Cysteine/Cystine |
| 10 mM/1 mM |
| Adsorption of denatured/reduced lysozyme on zeolite: An aliquot of zeolite (20 mg) was equilibrated with 500 μL of denaturing buffer for 10 min. Then, 500 μL of 4 mg/mL denatured/reduced lysozyme was added and incubated for varying amounts of time at room temperature on a Rotator RT-50 (Taitec Corporation, Saitama, Japan). The zeolite was removed by centrifugation and the protein concentration of the supernatant was measured. The amount of protein adsorbed was calculated by subtracting the amount of protein in the supernatant after centrifugation from the amount of lysozyme before adsorption. Refolding lysozyme: An aliquot of zeolite (50 mg) was equilibrated with 500 μL of denaturing buffer for 10 min. Then, 500 μL of denatured lysozyme solution (2 mg/mL) was added and incubated for 1 h at room temperature. The lysozyme-adsorbed zeolite samples were washed four times with 5 mM Tris–HCl (pH 7.5). The elution and refolding of lysozyme were initiated by incubating it with refolding buffer (50 mM Tris–HCl [pH 8.5], 500 mM NaCl, 5 mg/mL PEG 20000, 1 mM EDTA, 10 mM Cys, and 1 mM cystine) containing Gdn-HCl and/or Arg at 4 °C. The zeolite was removed by centrifugation and the lysozyme activity and concentration in the supernatant were measured. All experiments were repeated at least three times. |
| Far-UV Circular Dichroism |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |